Gene Mosaicism Screening Using Single-Molecule Molecular Inversion Probes in Routine Diagnostics for Systemic...

Area of Science:

• Genetics
• Immunology
• Molecular Biology

Background:

• Systemic autoinflammatory diseases (SAIDs) pose diagnostic challenges, often leading to delayed treatment and organ damage.
• Postzygotic mutations, even at low allele frequencies, are implicated in some dominantly inherited SAIDs.
• Current next-generation sequencing methods struggle to differentiate low-grade mosaic variants from background noise.

Purpose of the Study:

• To develop a sensitive deep sequencing assay for accurate mosaicism detection in SAID-associated genes.
• To overcome the limitations of standard sequencing in identifying low-allele-frequency postzygotic mutations.

Main Methods:

• Development of a sensitive deep sequencing assay utilizing single-molecule molecular inversion probes.
• Application of the assay for mosaicism detection in genes associated with SAIDs.

Main Results:

• The assay accurately detects postzygotic variants with allele frequencies as low as 1%.
• The probability of correctly identifying mutations with allele frequencies ≥3% exceeds 99.9%.
• Three patients with mosaicism were identified, including two with likely pathogenic NLRP3 variants and one with a likely pathogenic TNFRSF1A variant (1.3% allele frequency).

Conclusions:

• The developed assay is a flexible, robust, fast, cost-effective, and reliable method for mosaicism detection in SAID-associated genes.
• This technology is well-suited for routine diagnostics, potentially improving early diagnosis and management of SAIDs.